The present invention relates to the field of protein purification. Specifically, the present invention relates to discovery of and isolation of affinity ligands useful for the purification of tissue plasminogm activator, or tPA.
Human tissue-type plasminogen activator, or tPA, is a proteolytic enzyme produced by endothelial cells which has high affinity for fibrin contained in aggregates of coagulated blood (i.e., clots, or xe2x80x9cthrombixe2x80x9d). tPA also serves to activate and convert plasminogen, an inactive proenzyme, into plasmin, a thrombolytic enzyme. Since tPA binds to fibrin in thrombi and activates plasminogen there to dissolve clots, tPA has become an important drug for use as a thrombolytic.
Although tPA has become a leading drug in the treatment of thrombosis, it competes against other effective thrombolytic agents, such as streptokinase and urokinase, which are arguably less effective but cost much less. In order for tPA to remain among the most-prescribed thrombolytic agents or to be distributed to even greater numbers of patients, ways in which tPA can be produced more efficiently or at lower cost must be explored.
Effective means for eliminating impurities such as cell debris, pathogens, non-human proteins, etc. from a production feed stream is also important in the production of tPA, as it is with any protein product intended ultimately for therapeutic administration to human patients.
Thus, there is a continuing need for the development of improved reagents, materials and techniques for the isolation of tPA on a more efficient and cost effective basis.
Affinity chromatography is a very powerful technique for achieving dramatic single-step increases in purity. Narayanan (1994), for instance, reported a 3000-fold increase in purity through a single affinity chromatography step.
Affinity chromatography is not, however, a commonly used technique in large-scale production of biomolecules. The ideal affinity chromatography ligand must, at acceptable cost, (1) capture the target biomolecule with high affinity, high capacity, high specificity, and high selectivity; (2) either not capture or allow differential elution of other species (impurities); (3) allow controlled release of the target under conditions that preserve (i.e., do not degrade or denature) the target; (4) permit sanitization and reuse of the chromatography matrix; and (5) permit elimination or inactivation of any pathogens. However, finding high-affinity ligands of acceptable cost that can tolerate the cleaning and sanitization protocols required in pharmaceutical manufacturing has proved difficult (see, Knight, 1990).
Murine monoclonal antibodies (MAbs) have been used effectively as affinity ligands. Monoclonal antibodies, on the other hand, are expensive to produce, and they are prone to leaching and degradation under the cleaning and sanitization procedures associated with purification of biomolecules, leading MAb-based affinity matrices to lose activity quickly (see, Narayanan, 1994; Boschetti, 1994). In addition, although MAbs can be highly specific for a target, the specificity is often not sufficient to avoid capture of impurities that are closely related to the target. Moreover, the binding characteristics of MAbs are determined by the immunoglobulin repertoire of the immunized animal, and therefore practitioners must settle for the binding characteristics they are dealt by the animal""s immune systte i.e., there is little opportunity to optimize or select for particular binding or elution characteristics using only MAb technology. Finally, the molecular mass per binding site (25 kDa to 75 kDa) of MAbs and even MAb fragments is quite high.
Up until now, there have been no known affinity ligands suitable for the purification of tPA that approach the characteristics of the ideal affinity ligand described above, that not only bind to the target tPA molecule with high affinity but also release the tPA under desirable or selected conditions, that are able to discriminate between the tPA and other components of the solution in which the tPA is presented, and/or that are able to endure cleaning and sanitization procedures to provide regenerable, reusable chromatogaphic matrices.
Such tPA affinity ligands and methods for obtaining them are provided herein.
The present invention provides methods for obtaining affinity ligands for tPA which exhibit desirable or selected binding properties and release properties. The affinity ligands of the present invention exhibit not only favorable binding characteristics for affinity separation of tPA but also desired release (elution) characteristics and other desired properties such as stability, resistance to degradation, durability, reusability, and ease of manufacture.
The tPA affinity ligands of the present invention may be initially identified from a peptide library, such as a phage display library, by a method comprising:
(a) selecting a first solution condition (i.e., the binding conditions) at which it is desired that an affinity ligand should bind to the tPA;
(b) selecting a second solution condition (i.e., the release conditions) at which it is desired that an affinity complex between the tPA and the affinity ligand will dissociate, wherein the second solution condition is different from the first solution condition;
(c) providing a library of analogues of a candidate binding domain, wherein each analogue differs from said candidate binding domain by variation of the amino acid sequence at one or more amino acid positions within the domain;
(d) contacting said library of analogues with tPA at the first solution condition, for sufficient time to permit analogue/tPA binding complexes to form;
(e) removing analogues that do not bind under the first solution condition;
(f) altering the conditions of the solution of contacting step (e) to the second solution condition; and
(g) recovering the candidate binding analogues released under the second solution condition, wherein the recovered analogues identify isolated tPA affinity ligands.
Following this general procedure, several polypeptide affinity ligands for tPA have been isolated, as described in more detail below.